CVC與真菌生物被膜

CVC相關(guān)性念珠菌感染與治療策略山東大學(xué)齊魯醫(yī)院重癥醫(yī)學(xué)科丁士163.com內(nèi)容念珠菌生物被膜特點(diǎn)及危害CVC相關(guān)念珠菌感染與棘白菌素藥物治療優(yōu)勢(shì)CVC感染抗真菌治療策略形成生物被膜是念珠菌侵襲性感染的前提FungalBiofilmsandDrugResistance.EmergingInfectiousDiseases?/eid?Vol.10,No.1,January2004FungalBiofilms:RelevanceintheSettingofHumanDisease.CurrFungalInfectRep.2010December1;4(4):266–275.產(chǎn)生物被膜的念珠菌菌株附著有機(jī)玻璃或硅膠片，在0.05%(v/v)

Calcofluor-White浸染1min染料特異性與真菌細(xì)胞壁中的幾丁質(zhì)和葡聚糖特異性結(jié)合熒光顯微鏡下觀察早期(0to11h),微小菌落中期(12to30h),類似細(xì)胞壁構(gòu)成無定形物質(zhì)覆蓋菌落成熟期(38to72h),菌落被覆蓋念珠菌生物被膜相關(guān)感染隨時(shí)間延長，形成類似生物被膜白色念珠菌皮膚感染模型生根發(fā)芽，咬定青山不松口ThePathogenesisofCandidaInfectionsinaHumanSkinModel:ScanningElectronMicroscopeObservations.ISRNDermatol.2011;2011:150642Candidaalbicans-EndothelialCellInteractions:aKeyStepinthePathogenesisofSystemicCandidiasis.INFECTIONANDIMMUNITY,2008,76(10):4370–4377侵襲性念珠菌感染與生物被膜NIHinPA-03-047報(bào)道，80%微生物感染與生物被膜形成相關(guān)生物被膜是指細(xì)(真)菌吸附于機(jī)體腔道或生物材料表面，分泌多糖基質(zhì)、纖維蛋白等多種細(xì)胞外基質(zhì)成分，將自身包繞其中形成的膜樣多細(xì)(真)菌復(fù)合體生物被膜內(nèi)殘存的細(xì)(真)菌不斷外逸，形成新的感染灶，引起感染持續(xù)存在，導(dǎo)致感染被徹底治愈機(jī)會(huì)減少，臨床治療失敗PLoSONE.2009,4(11):e7967MICROBIOLOGYANDMOLECULARBIOLOGYREVIEWS,2000,64(4):847–867Transcription.2012Nov-Dec;3(6):315-22.NatRevMicrobiol.2011February;9(2):109–118(臭名昭著)鋼筋混凝土構(gòu)成的社區(qū)群體產(chǎn)生物被膜念珠菌的超微結(jié)構(gòu)

beta-1,3

glucan的作用形成生物被膜過程中，念珠菌形態(tài)改變生物被膜念珠菌細(xì)胞壁厚度為浮游念珠菌的2倍PutativeRoleof-1,3GlucansinCandidaalbicansBiofilmResistance.ANTIMICROBIALAGENTSANDCHEMOTHERAPY,2007,51(2):510–520形成生物被膜念珠菌細(xì)胞壁β-1,3glucan含量顯著高于靜止期和對(duì)數(shù)生長期念珠菌(P<0.001)由碳水化合物約占白色念珠菌細(xì)胞壁80%-90%β-1,3andβ-1,6glucans(50to60%),mannoproteins(30to40%),andchitin(0.6to9%)beta-glucan在產(chǎn)生物被膜念珠菌的作用生物被膜念珠菌合成、釋放更多β-1,3glucan示意圖ACandidaBiofilm-InducedPathwayforMatrixGlucanDelivery:ImplicationsforDrugResistance.PLoSPathog8(8):e1002848β-D-glucan在念珠菌侵襲感染的診斷價(jià)值Findingthe“missing50%”ofinvasivecandidiasis:hownonculturediagnosticswillimproveunderstandingofdiseasespectrumandtransformpatientcare.

ClinInfectDis.2013May;56(9):1284-92碳水化合物約占白色念珠菌細(xì)胞壁80%-90%β-1,3andβ-1,6glucans(50to60%),mannoproteins(30to40%),andchitin(0.6to9%)8念珠菌生物被膜模型FungalBiofilmsandDrugResistance.EmergingInfectiousDiseases?/eid?Vol.10,No.1,January2004EffectofGrowthRateonResistanceofCandidaalbicansBiofilmstoAntifungalAgents.ANTIMICROBIALAGENTSANDCHEMOTHERAPY,1998,1900–190542(8)FungalBiofilms:RelevanceintheSettingofHumanDisease.CurrFungalInfectRep.2010December1;4(4):266–275.InterfaceofCandidaalbicansBiofilmMatrix-AssociatedDrugResistanceandCellWallIntegrityRegulationSEM顯示白色念珠菌在多孔濾網(wǎng)形成生物被膜，顯示孢子和菌絲被細(xì)胞外基質(zhì)包繞大鼠白色念珠菌相關(guān)CVC感染模型(24h)顯示酵母細(xì)胞與菌絲倍細(xì)胞外基質(zhì)覆蓋熱帶念珠菌生物被膜模型（48h模型）白色念珠菌在硅膠板形成生物被膜模型（流動(dòng)狀態(tài)）敏感與耐藥念珠菌生物被膜存在差異CO23：氟康唑敏感白色念珠菌CO23RFK：米卡芬凈耐藥白色念珠菌CO23RFLC：氟康唑耐藥白色念珠菌念珠菌敏感菌株生物被膜模型：起始階段粘附孢子少，結(jié)構(gòu)疏松，菌絲短耐藥菌株生物被膜模型：大量孢子和菌絲構(gòu)成；CO23RFK富含大量孢子和短菌絲；CO23RFLC以長菌絲為主

Characterizationof

biofilms

indrug-sensitiveanddrug-resistantstrainsof

Candida.JChemother.

2013Apr;25(2):87-95念珠菌耐抗真菌藥物機(jī)制Astickysituation:

untangling

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transcriptional

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controlling

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in

Candida

albicans.

Transcription.

2012;3(6):315-22.TRENDSinMicrobiologyVol.11No.1January2003NatRevMicrobiol.2011February;9(2):109–118金剛罩！鐵布衫！反導(dǎo)系統(tǒng)！鋼筋混凝土構(gòu)成的群體，阻擋藥物穿透念珠菌生物被膜影響氟康唑分布

念珠菌耐抗真菌藥物機(jī)制B絕大部分氟康唑分布在非白色念珠菌生物被膜、非白色念珠菌細(xì)胞壁或細(xì)胞漿內(nèi)極少或無氟康唑分布生物被膜念珠菌細(xì)胞壁結(jié)合氟康唑是浮游念珠菌的4~5倍，意味相當(dāng)一部分氟康唑分布在生物被膜和細(xì)胞壁，不能進(jìn)入細(xì)胞漿，增加念珠菌耐藥性Roleofmatrixglucaninantifungalresistanceofnon-albicanscandidabiofilms.AntimicrobAgentsChemother.2013Apr;57(4):1918-20生物被膜念珠菌細(xì)胞膜固醇成分改變生物被膜與浮游白念珠菌麥角固醇水平在6h時(shí)相同，生物膜成熟期減少50%，而浮游細(xì)胞在6～12h減少18%，其他固醇水平在兩者之間也有明顯差異麥角固醇比例改變影響抗真菌藥物進(jìn)入念珠菌通透性，進(jìn)而防止或阻滯抗真菌藥物進(jìn)入念珠菌細(xì)胞壁改變固醇成分比例影響生物被膜內(nèi)念珠菌對(duì)氟康唑耐藥性MechanismofFluconazoleResistanceinCandidaalbicansBiofilms:Phase-SpecificRoleofEffluxPumpsandMembraneSterols.InfectImmun.2003August;71(8):4333–4340.生物被摸形成與念珠菌耐藥MechanismsofCandidabiofilmdrugresistance.FutureMicrobiol.;8(10):10.2217/fmb.13.101.抗真菌藥物對(duì)生物被膜念珠菌療效兩性霉素B對(duì)生物被膜念珠菌MIC增加脂質(zhì)體兩性霉素B對(duì)生物被膜念珠菌MIC無顯著變化氟康唑和伏立康唑?qū)Ω∮文钪榫鶰IC低，對(duì)生物被膜念珠菌MIC極高與浮游念珠菌比較，生物被膜念珠菌對(duì)氟康唑耐藥性高達(dá)1000倍米卡芬凈和卡泊芬凈對(duì)浮游和生物被膜念珠菌MIC無顯著差異MICsinCandidabiofilmsincrease100–1000timescomparedwithplanktoniccellsAntifungalSusceptibilityofCandidaBiofilms:UniqueEfficacyofAmphotericinBLipidFormulationsandEchinocandins.ANTIMICROBIALAGENTSANDCHEMOTHERAPY,June2002,p.1773–1780Vol.46,No.6ActivitiesofFluconazole,Caspofungin,Anidulafungin,andAmphotericinBonPlanktonicandBiofilmCandidaSpeciesDeterminedbyMicrocalorimetry。AntimicrobAgentsChemother.2014May;58(5):2709–2717重癥患者侵襲性念珠菌感染：生理屏障破壞Revisitingthesourceofcandidemia:skinorgut?ClinInfectDis2001;33:1959–67.侵襲性念珠菌感染常由腸道念珠菌大量繁殖進(jìn)入血流所致最易受累：腎、心、腦、肺16重癥患者侵襲性念珠菌感染

危險(xiǎn)因素：解剖生理屏障完整性破壞

ICU患者最突出特點(diǎn)是其解剖生理屏障完整性破壞,定植體表皮膚和體腔粘膜表面的條件致病真菌，以及環(huán)境中真菌侵入原本無菌深部組織和血液侵襲性念珠菌感染常由正常腸道念珠菌大量繁殖進(jìn)入血流所致。最易受累：腎、心、腦、肺AmousemodelforCandidaglabratahematogenousdisseminatedinfectionstartingfromthegut:evaluationofstrainswithdifferentadhesionproperties.PLoSOne.2013Jul23;8(7):e69664.DisruptionoftheintestinalmucosalbarrierinCandidaalbicansinfections.MicrobiolRes.2013Aug25;168(7):389-95.NucciM,AnaissieE.Revisitingthesourceofcandidemia:skinorgut?ClinInfectDis2001;33:1959–67.股靜脈置管ICU院感約20%為血流感染(BSI)，87%與中心靜脈導(dǎo)管(CVC)有關(guān)白色念珠菌占血管內(nèi)導(dǎo)管分離病原體第二位中心靜脈導(dǎo)管相關(guān)真菌血癥與生物膜(A)×450;

(B)×1100;(C)×4500.由塔狀或蘑菇狀微菌落組成，其余空間被網(wǎng)狀分布的胞外多聚基質(zhì)所占據(jù)一張牢不可破的網(wǎng)AssessmentofthetypesofcatheterinfectivitycausedbyCandidaspeciesandtheirbiofilmformation.FirststudyinanintensivecareunitinAlgeria.IntJGenMed.2013;6:1–7.

×35NettJetal.Antimicrob.AgentsChemother.2007;51:510-520破壞生物被膜有助改善氟康唑療效超大劑量氟康唑(為浮游念珠菌MIC1000倍)對(duì)生物被膜念珠菌感染無效大劑量β-1,3glucanase能破壞念珠菌生物被膜小劑量β-1,3glucanase不能破壞念珠菌生物被膜小劑量β-1,3glucanase聯(lián)合超大劑量氟康唑，清除念珠菌生物被膜伏立康唑治療白色念珠菌感染性心內(nèi)膜炎模型療效差(A)對(duì)照組72h;(B)氟咆嘧啶24h;(C)伏立康唑72h，作用有限；(D)米卡芬靜48h，顯著破壞念珠菌結(jié)構(gòu)ActivitiesandUltrastructuralEffectsofAntifungalCombinationsagainstSimulatedCandidaEndocardialVegetations.ANTIMICROBIALAGENTSANDCHEMOTHERAPY,July2008,p.2367–2376Vol.52,No.7兩性霉素B與氟康唑

不能抑制生物被膜念珠菌生長C.kruseiATCC6258C.parapsilosisATCC22019C.albicansHK1Sa(A)Control(B)exposedto600ug/mlamphotericinBfor4h(C)exposedto600ug/mlfluconazolefor4hThewrinkled,shrunk,ruptured,andballooningeffectofthedrugonyeastcellsInVitroMethodToStudyAntifungalPerfusioninCandidaBiofilms.JOURNALOFCLINICALMICROBIOLOGY,2005,43(2):818–825兩性霉素B脂質(zhì)體抑制生物被膜念珠菌生長RabbitModelofCandidaalbicansBiofilmInfection:LiposomalAmphotericinBAntifungalLockTherapy.ANTIMICROBIALAGENTSANDCHEMOTHERAPY,2004,48(5):1727–17327d兔頸靜脈生物膜模型3d兔頸靜脈生物膜模型對(duì)照兩性霉素B脂質(zhì)體1mg/100ul氟康唑1mg/100ul念珠菌感染兔CVC模型，每天導(dǎo)管內(nèi)局部灌注8h，連續(xù)3天或7天氟康唑生物被膜有所減少，念珠菌感染部位生物被膜形態(tài)與對(duì)照組相似(念珠菌生長)脂質(zhì)體兩性霉素B幾乎完全清除CVC表面生物被膜相關(guān)念珠菌，1~2處殘存感染部位缺乏生物被膜(無念珠菌生長)脂質(zhì)體兩性霉素B有效治療生物被膜念珠菌感染，但機(jī)制不明，脂質(zhì)體無真菌抑制作用棘白菌素類藥物作用機(jī)制Stress,

drugs,and

evolution:the

role

of

cellular

signaling

in

fungal

drugresistance.EukaryotCell.2008;7(5):747-64.Resistancetoechinocandin-classantifungaldrugs.DrugResistUpdat.2007June;10(3):121–130.Stress,

drugs,and

evolution:the

role

of

cellular

signaling

in

fungal

drugresistance.EukaryotCell.2008;7(5):747-64.Resistancetoechinocandin-classantifungaldrugs.DrugResistUpdat.2007June;10(3):121–130.Fungalechinocandinresistance.FungalGenetBiol.

2010;47(2):117-26.ChoiHWetal.AntimicrobAgentsChemother2007;51:1520-23棘白菌素類抗真菌藥物作用靶點(diǎn)為真菌細(xì)胞壁β-1.3-葡聚糖，生物被膜基質(zhì)中含有β-1.3-葡聚糖通過減少、抑制β-葡聚糖產(chǎn)生，破壞生物被膜完整性，有利控制念珠菌生物被膜感染卡泊芬凈抑制生物被膜念珠菌存活I(lǐng)nVitroActivityofCaspofunginagainstCandidaalbicansBiofilms.ANTIMICROBIALAGENTSANDCHEMOTHERAPY.2002,46(11):3591–3596卡泊芬凈治療組(0.5ug/ml)生物被膜內(nèi)念珠菌菌絲少、孢子形態(tài)異常未治療組，存在大量活性代謝念珠菌(從綠色到紅色，以紅色為主)；卡泊芬凈治療組(0.5ug/ml)，為彌漫性綠色，提示存在大量無代謝活性(死亡)念珠菌棘白菌素完全抑制白色念珠菌形成生物被膜Real-TimeMicroscopicObservationofCandidaBiofilmDevelopmentandEffectsDuetoMicafunginandFluconazole.

AntimicrobAgentsChemother.2013May;57(5):2226-2230.棘白菌素類藥物治療重癥患者念珠菌感染優(yōu)勢(shì)5,(6)-carboxyfluoresceindiacetate(CFDA),羧基熒光素二醋酸酯5(6)-CFDA是膜透性染料，可通過孵化進(jìn)入細(xì)胞。一旦進(jìn)入細(xì)胞后，CFDA被細(xì)胞內(nèi)酯酶水解形成羧基熒光素bis-(1,3-dibutylbarbituricacid)trimethineoxonol(DiBAC),DiBAC4(3)是一種檢測(cè)細(xì)胞膜電位的親脂性陰離子熒光染料，它本身無熒光，當(dāng)進(jìn)人細(xì)胞與胞漿內(nèi)的蛋白質(zhì)結(jié)合后才發(fā)出熒光。DiBAC4(3)進(jìn)入細(xì)胞，細(xì)胞內(nèi)熒光強(qiáng)度增加，即膜電位增加表示細(xì)胞去極化;反之，細(xì)胞內(nèi)熒光強(qiáng)度降低即膜電位降低表示細(xì)胞超極化TheAntifungalEchinocandinCaspofunginAcetateKillsGrowingCellsofAspergillusfumigatusInVitro.ANTIMICROBIALAGENTSANDCHEMOTHERAPY,Sept.2002,p.3001–3012Vol.46,No.9棘白菌素類藥物治療優(yōu)勢(shì)Anti-biofilmactivityRapidfungicidalactivity治療侵襲性念珠菌感染，初始治療選用殺菌作用的抗真菌藥物UnchangedactivityagainstCandidaspp.ShowingdecreasedsusceptibilitytofluconazoleandtootherazolesAnti-cytokineandanti-chemokineactivity抗真菌藥物藥代動(dòng)力學(xué)-藥效動(dòng)力學(xué)Pharmacokinetic–pharmacodynamicoptimizationoftriazoleantifungaltherapy.CurrOpinInfectDis24(suppl2):S14–S29CritCareMed2013;41:580–637InternationalJournalofAntimicrobialAgents39(2012)1–102003，2008，2012膿毒癥指南：初始經(jīng)驗(yàn)性抗感染治療包括一種或多種對(duì)可能致病菌(細(xì)菌，和/或真菌，或病毒)敏感，且以足夠藥物濃度抵達(dá)導(dǎo)致膿毒癥的感染部位的藥物(I-B)棘白菌素類藥物改善念珠菌血癥患者預(yù)后Riskfactorsandoutcomesofcandidemiacausedbybiofilm-formingisolatesinatertiarycarehospital.PLoSOne.2012;7(3):e33705.

2005-2007，84例為形成生物被膜念珠菌血癥，123例為不能形成生物被膜念珠菌血癥。30天生存率不同(p=0.004)生物被膜組無生物被膜組Pvalue住院死亡率51.2%(43/84)31.7%(39/123)0.004感染相關(guān)死亡率44.1%(37/84)27.6%(34/123)0.01早期(<24h)充分抗真菌治療與拔出CVC改善念珠菌血癥導(dǎo)致休克患者預(yù)后SepticShockAttributedtoCandidaInfection:ImportanceofEmpiricTherapyandSourceControl.ClinicalInfectiousDiseases2012;54(12):1739–46180例念珠菌血癥休克患者，24h內(nèi)充分抗真菌治療和充分控制感染灶(如：拔出CVC、外科或置管引流)，患者死亡率52.8%,非目標(biāo)治療死亡率97.6%(P<0.001)氟康唑：非光滑念珠菌6mg/kg，光滑念珠菌12mg/kg；LP-AMB：≥3mg/kg；卡泊芬凈：70/50mg發(fā)生休克24內(nèi)治療發(fā)生休克24內(nèi)未治療52.897.62012ESCMID非粒缺成人患者

侵襲性念珠菌感染指南血培養(yǎng)酵母菌陽性(AII)或經(jīng)驗(yàn)治療(CIIu)開始抗真菌治療Stronglyrecommended:棘白菌素(A-I)Moderatelyrecommended:L-AMBor伏立康唑(B-I)Marginallyrecommended:氟康唑orABLC(C-I)recommendationagainstuse(D):AMB伊曲康唑泊沙康唑聯(lián)合治療ClinMicrobiolInfect2012;18(Suppl.7):1–8ClinMicrobiolInfect2012;18(Suppl.7):9–18ClinMicrobiolInfect2012;18(Suppl.7):19–37u-uncontrolledtrials2009IDSA2012ESCMID氟康唑A-IlesscriticallyillandwhohavenorecentazoleexposureA-IIIC-I棘白菌素類藥物A-Imoderatelyseveretosevereillness&recentazoleexposureA-IIIA-I伏立康唑A-I(alternativeagent)B-I兩性霉素B脂質(zhì)體A-I(alternativeagents)B-I兩性霉素B傳統(tǒng)劑型A-I(alternativeagent)D-I一枝獨(dú)秀，一落千丈，打入冷宮2012ESCMID指南

念珠菌血癥患者管理與療程建議每日至少一次血培養(yǎng)，直至第一次念珠菌血培養(yǎng)陰性。第一次念珠菌血培養(yǎng)陰性后，繼續(xù)抗真菌治療14天念珠菌對(duì)氟康唑敏感，靜脈針劑治療10天后病情穩(wěn)定，降階梯治療ESCMID*guidelineforthediagnosisandmanagementofCandidadiseases2012:non-neutropenicadultpatients.ClinMicrobiolInfect2012;18(Suppl.7):19–37及早拔除CVC改善念珠菌血癥預(yù)后嗎單因素分析及早拔除CVC改善28d和42d生存率，不改善念珠菌血癥清除率Earlyremovalofcentralvenouscatheterinpatientswithcandidemiadoesnotimproveoutcome:analysisof842patientsfrom2randomizedclinicaltrials.ClinInfectDis.2010Aug1;51(3):295-303.疾病嚴(yán)重程度和拔除CVC對(duì)死亡率影響多因素分析未顯示拔除CVC保護(hù)作用APACHEII、高齡、持續(xù)粒細(xì)胞缺乏與死亡率相關(guān)Earlyremovalofcentralvenouscatheterinpatientswithcandidemiadoesnotimproveoutcome:analysisof842patientsfrom2randomizedclinicaltrials.ClinInfectDis.2010Aug1;51(3):295-303.及早拔除CVC改善念珠菌血癥預(yù)后嗎米卡芬凈100

mg/d

vs兩性霉素B脂質(zhì)體3

mg/kg/d

米卡芬凈100

mg/d，米卡芬凈150

mg/d

vs卡泊芬凈70/50

mg/dEarlyremovalofcentralvenouscatheterinpatientswithcandidemiadoesnotimproveoutcome:analysisof842patientsfrom2randomizedclinicaltrials.ClinInfectDis.2010Aug1;51(3):295-303.棘白菌素類藥物局部封閉不能完全抑制生物被膜念珠菌感染Candidabiofilmsaged12h(youngbiofilm)and5days(maturebiofilm)PossibleroleofazoleandechinocandinlocksolutionsinthecontrolofCandidabiofilmsassociat